Biomedical subjects

Pierre Lepage

Publications and source records attributed to Pierre Lepage.

16 recordsLinked to original sources

PubMed2006

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Acetyltransferases↗

PubMed2006

Identification of CD109 as part of the TGF-beta receptor system in human keratinocytes.

We have previously reported that keratinocytes defective in glycosylphosphatidylinositol (GPI)-anchor biosynthesis display enhanced TGF-beta responses. These studies implicated the involvement of a 150 kDa GPI-anchored TGF-beta1 binding protein, r150, in modulating TGF-beta signaling. Here, we sought to determine the molecular identity of r150 by affinity purification and microsequencing. Our results identify r150 as CD109, a novel member of the alpha2-macroglobulin (alpha2M)/complement superfamily, whose function has remained obscure. In addition, we have identified a novel CD109 isoform that occurs in the human placenta but not keratinocytes. Biochemical studies show that r150 contains an internal thioester bond, a defining feature of the alpha2M/complement family. Loss and gain of function studies demonstrate that CD109 is a component of the TGF-beta receptor system, and a negative modulator of TGF-beta responses in keratinocytes, as implicated for r150. Our data suggest that CD109 can inhibit TGF-beta signaling independently of ligand sequestration and may exert its effect on TGF-beta signaling by direct modulation of receptor activity. Together, our results linking CD109 function to regulation of TGF-beta signaling suggest that CD109 plays a unique role in the regulation of isoform-specific TGF-beta signaling in keratinocytes.

Antigens, CD↗

PubMed2006

Mutation and biochemical analysis of patients belonging to the cblB complementation class of vitamin B12-dependent methylmalonic aciduria.

Methylmalonic aciduria, cblB type (OMIM 251110) is an inborn error of vitamin B(12) metabolism that occurs due to mutations in the MMAB gene. MMAB encodes the enzyme ATP:cobalamin adenosyltransferase, which catalyzes the synthesis of the coenzyme adenosylcobalamin required for the activity of the mitochondrial enzyme methylmalonyl CoA mutase (MCM). MCM catalyzes the isomerization of methylmalonyl CoA to succinyl CoA. Deficient MCM activity results in methylmalonic aciduria and a susceptibility to life-threatening acidotic crises. The MMAB gene was sequenced from genomic DNA from a panel of 35 cblB patients, including five patients previously investigated. Nineteen MMAB mutations were identified, including 13 previously unknown mutations. These included 11 missense mutations, two duplications, one deletion, four splice-site mutations, and one nonsense mutation. None of these mutations was identified in 100 control alleles. Most of the missense mutations (9/11) were clustered in exon 7; many of these affected amino acid residues that are part of the probable active site of the enzyme. One previously described mutation, c.556C >T (p.R186W), was particularly common, accounting for 33% of pathogenic alleles. It was seen almost exclusively in patients of European background and was typically associated with presentation in the first year of life.

Alkyl and Aryl Transferases↗

PubMed2006

Spectrum of mutations in mut methylmalonic acidemia and identification of a common Hispanic mutation and haplotype.

Cobalamin nonresponsive methylmalonic acidemia (MMA, mut complementation class) results from mutations in the nuclear gene MUT, which codes for the mitochondrial enzyme methylmalonyl CoA mutase (MCM). To better elucidate the spectrum of mutations that cause MMA, the MUT gene was sequenced in 160 patients with mut MMA. Sequence analysis identified mutations in 96% of disease alleles. Mutations were found in all coding exons, but predominantly in exons 2, 3, 6, and 11. A total of 116 different mutations, 68 of which were novel, were identified. Of the 116 different mutations, 53% were missense mutations, 22% were deletions, duplications or insertions, 16% were nonsense mutations, and 9% were splice-site mutations. Sixty-one of the mutations have only been identified in one family. A novel mutation in exon 2, c.322C>T (p.R108C), was identified in 16 of 27 Hispanic patients. SNP genotyping data demonstrated that Hispanic patients with this mutation share a common haplotype. Three other mutations were seen exclusively in Hispanic patients: c.280G>A (p.G94R), c.1022dupA, and c.970G>A (p.A324T). Seven mutations were seen almost exclusively in black patients, including the previously reported c.2150G>T (p.G717V) mutation, which was identified in 12 of 29 black patients. Two mutations were seen only in Asian patients. Some frequently identified mutations were not population-specific and were identified in patients of various ethnic backgrounds. Some of these mutations were found in mutation clusters in exons 2, 3, 6, and 11, suggesting a recurrent mutation.

Alleles↗

PubMed2006

Common polymorphisms in the promoter of the visfatin gene (PBEF1) influence plasma insulin levels in a French-Canadian population.

The adipokine visfatin (PBEF1) exhibits insulin-mimetic effects and correlates strongly with visceral adiposity. We sequenced visfatin gene exons and 1,480 bp of the promoter in 23 individuals, including 18 individuals from the Quebec Family Study (QFS) with varying degrees of abdominal visceral fat, assessed by computed tomography, and 5 individuals from the Saguenay-Lac-Saint-Jean region of Québec. We identified a synonymous polymorphism in exon 7 (SER301SER) but no nonsynonymous mutations. We observed an additional 10 polymorphisms, including 5 intronic, 4 within the promoter, and 1 within the 3' untranslated region. Further promoter sequencing (816 bp) identified five additional single nucleotide polymorphisms (SNPs) in the QFS population. To investigate the role of visfatin gene variants in obesity-related phenotypes, we genotyped a total of 13 SNPs in the promoter region of the gene. From these, we analyzed the seven common SNPs in the QFS sample (918 participants from 208 families). A significant association was found between two SNPs (rs9770242 and rs1319501), in perfect linkage disequilibrium, and fasting insulin levels (P = 0.002). These SNPs were also associated with fasting glucose (P <or= 0.02). In addition, a more distal SNP (rs7789066) was significantly associated with the apolipoprotein B component of VLDL (P = 0.012).

Adult↗

PubMed2005

Functional organization of a Schwann cell enhancer.

Myelin basic protein (MBP) gene expression is conferred in oligodendrocytes and Schwann cells by different upstream enhancers. In Schwann cells, expression is controlled by a 422 bp enhancer lying -9 kb from the gene. We show here that it contains 22 mammalian conserved motifs > or =6 bp. To investigate their functional significance, different combinations of wild-type or mutated motifs were introduced into reporter constructs that were inserted in single copy at a common hypoxanthine phosphoribosyltransferase docking site in embryonic stem cells. Lines of transgenic mice were derived, and the subsequent qualitative and quantitative expression phenotypes were compared at different stages of maturation. In the enhancer core, seven contiguous motifs cooperate to confer Schwann cell specificity while different combinations of flanking motifs engage, at different stages of Schwann cell maturation, to modulate expression level. Mutation of a Krox-20 binding site reduces the level of reporter expression, whereas mutation of a potential Sox element silences reporter expression. This potential Sox motif was also found conserved in other Schwann cell enhancers, suggesting that it contributes widely to regulatory function. These results demonstrate a close relationship between phylogenetic footprints and regulatory function and suggest a general model of enhancer organization. Finally, this investigation demonstrates that in vivo functional analysis, supported by controlled transgenesis, can be a robust complement to molecular and bioinformatics approaches to regulatory mechanisms.

Amino Acid Motifs↗

PubMed2005

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Amino Acid Sequence↗

PubMed2005

Mapping common regulatory variants to human haplotypes.

Inter-individual variation in gene expression has proven to be in part governed by genetic determinants, which may be trans- or cis-acting. The underlying cause of cis-acting regulatory variation has been identified in only a handful of the hundreds of genes shown to display differential allelic expression. In this report, we describe a systematic effort to map common cis-acting variants in 64 genes, using association methods in HapMap samples. We identified 16 loci (25%), each of which harbors common haplotypes that affect total expression of a gene, and a further 17 loci (27%) with evidence of haplotypes affecting relative allelic expression in heterozygote samples. Our survey suggests that detailed mapping of allele-specific in vivo expression will provide a rich source of regulatory SNPs or haplotypes that should be given high priority in association studies of human phenotypes.

Chromosome Mapping↗

PubMed2005

Survey of allelic expression using EST mining.

Cis-acting allelic variation in gene regulation is a source of phenotypic variation. Consequently, recent studies have experimentally screened human genes in an attempt to initiate a catalog of genes possessing cis-acting variants. In this study, we use human EST data in dbEST as the source of allelic expression data, and the HapMap database to provide expected allele frequencies in human populations. We demonstrate a greater concordance of allele frequencies estimated from human ESTs in dbEST with those derived from the CEPH HapMap sample representing Caucasians from northern and western Europe, than population samples obtained in Asia and Africa. Deviations between allele frequencies observed in EST databases and the ones obtained from the CEPH HapMap samples may result from common heritable cis-acting variants altering the relative allele distribution in RNA. We provide in silico as well as experimental evidence that this strategy does allow significant enrichment of genes harboring common heritable cis-acting polymorphisms in linkage disequilibrium with expressed alleles.

Alleles↗

PubMed2004

Susceptibility to leprosy is associated with PARK2 and PACRG.

Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year. It has long been thought that leprosy has a strong genetic component, and recently we mapped a leprosy susceptibility locus to chromosome 6 region q25-q26 (ref. 3). Here we investigate this region further by using a systematic association scan of the chromosomal interval most likely to harbour this leprosy susceptibility locus. In 197 Vietnamese families we found a significant association between leprosy and 17 markers located in a block of approx. 80 kilobases overlapping the 5' regulatory region shared by the Parkinson's disease gene PARK2 and the co-regulated gene PACRG. Possession of as few as two of the 17 risk alleles was highly predictive of leprosy. This was confirmed in a sample of 975 unrelated leprosy cases and controls from Brazil in whom the same alleles were strongly associated with leprosy. Variants in the regulatory region shared by PARK2 and PACRG therefore act as common risk factors for leprosy.

Alleles↗

PubMed2004

A survey of genetic and epigenetic variation affecting human gene expression.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or "allelic imbalances," can be observed in multiple samples derived from reference CEPH ("Centre d'Etude du Polymorphisme Humain") pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes (IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.

Allelic Imbalance↗

PubMed2004

Mutations in the MMAA gene in patients with the cblA disorder of vitamin B12 metabolism.

Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl-coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433C>T (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals.

Child, Preschool↗

PubMed2003

A combinatorial network of evolutionarily conserved myelin basic protein regulatory sequences confers distinct glial-specific phenotypes.

Myelin basic protein (MBP) is required for normal myelin compaction and is implicated in both experimental and human demyelinating diseases. In this study, as an initial step in defining the regulatory network controlling MBP transcription, we located and characterized the function of evolutionarily conserved regulatory sequences. Long-range human-mouse sequence comparison revealed over 1 kb of conserved noncoding MBP 5' flanking sequence distributed into four widely spaced modules ranging from 0.1 to 0.4 kb. We demonstrate first that a controlled strategy of transgenesis provides an effective means to assign and compare qualitative and quantitative in vivo regulatory programs. Using this strategy, single-copy reporter constructs, designed to evaluate the regulatory significance of modular and intermodular sequences, were introduced by homologous recombination into the mouse hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The proximal modules M1 and M2 confer comparatively low-level oligodendrocyte expression primarily limited to early postnatal development, whereas the upstream M3 confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. Mutation of putative Nkx6.2/Gtx sites within M3, although not eliminating oligodendrocyte targeting, significantly decreases transgene expression levels. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. In addition, when isolated from surrounding MBP sequences, M3 confers transient expression to Schwann cells elaborating myelin. These observations define the in vivo regulatory roles played by conserved noncoding MBP sequences and lead to a combinatorial model in which different regulatory modules are engaged during primary myelination, myelin maintenance, and remyelination.

Animals↗

PubMed2003

Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics.

Identifying the genes responsible for human diseases requires combining information about gene position with clues about biological function. The recent availability of whole-genome data sets of RNA and protein expression provides powerful new sources of functional insight. Here we illustrate how such data sets can expedite disease-gene discovery, by using them to identify the gene causing Leigh syndrome, French-Canadian type (LSFC, Online Mendelian Inheritance in Man no. 220111), a human cytochrome c oxidase deficiency that maps to chromosome 2p16-21. Using four public RNA expression data sets, we assigned to all human genes a "score" reflecting their similarity in RNA-expression profiles to known mitochondrial genes. Using a large survey of organellar proteomics, we similarly classified human genes according to the likelihood of their protein product being associated with the mitochondrion. By intersecting this information with the relevant genomic region, we identified a single clear candidate gene, LRPPRC. Resequencing identified two mutations on two independent haplotypes, providing definitive genetic proof that LRPPRC indeed causes LSFC. LRPPRC encodes an mRNA-binding protein likely involved with mtDNA transcript processing, suggesting an additional mechanism of mitochondrial pathophysiology. Similar strategies to integrate diverse genomic information can be applied likewise to other disease pathways and will become increasingly powerful with the growing wealth of diverse, functional genomics data.

Amino Acid Sequence↗

PubMed2002

Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency.

The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.

Animals↗

PubMed2002

5' flanking variants of resistin are associated with obesity.

Diabetes and obesity have long been known to be related. The recently characterized adipocyte hormone resistin (also called FIZZ3/ADSF) has been implicated as a molecular link between impaired glucose tolerance (IGT) and obesity in mice. A search for sequence variants at the human resistin locus identified nine single-nucleotide polymorphisms (SNPs) but no coding variants. An investigation into the association of these SNPs with diabetes and obesity revealed two 5' flanking variants (g.-537 and g.-420), in strong linkage disequilibrium, that are associated with BMI. In nondiabetic individuals from the Quebec City area and the Saguenay-Lac-St-Jean region of Quebec, the g.-537 mutation (allelic frequency = 0.04) was significantly associated with an increase in BMI (P = 0.03 and P = 0.01, respectively). When the data from these two populations were combined and adjusted for age and sex, both the g.-537 (odds ratio [OR] 2.72, 95% CI 1.28-5.81) and the g.-420 variants (1.58, 1.06-2.35) were associated with an increased risk for a BMI > or =30 kg/m(2). In contrast, in case/control and family-based study populations from Scandinavia, we saw no effect on BMI with either of these promoter variants. No association was seen with diabetes in any of the population samples.

5' Untranslated Regions↗